Progress 08/17/03 to 04/13/08
Outputs
Progress Report Objectives (from AD-416) Broaden the genetic base for upland cotton improvement. Develop pest resistant germplasm, breeding methodologies and marker assisted selection. Identify genes and gene products correlated with resistance to nematodes. Develop and use PCR based DNA markers for improvement of upland cotton. Approach (from AD-416) Broaden the germplasm base of upland cotton and develop special breeding populations by (1) accessing genetic diversity in exotic photoperiodic accessions, (2) developing elite breeding populations through random mating, (3) developing backcrossed chromosome substitution lines, and (4) developing chromosome specific recombinant inbred lines for selected G. barbadense backcrossed chromosome substitution lines. Develop pest resistant germplasm, breeding methodologies, and agronomic and fiber evaluations by (1) evaluating for agronomic and fiber properties and genetic analyses, (2) evaluating lines for tobacco budworm tolerance or resistance, (3) evaluating industry developed transgenic insect resistant lines to provide a non-industry view on performance of specific transgenes and transformation events, (4) discovering and developing root- knot and reniform nematode resistant germplasm, (5) breeding methodologies for root-knot nematode resistance, and (6) conducting evaluations in a regional root-knot nematode nursery. Identify genes and gene products correlated with resistance to nematodes by (1) developing hairy root system in cotton, and (2) characterizing MIC genes. Develop and utilize PCR based DNA markers for improvement of upland cotton by (1) developing informative PCR based EST-SSR markers, (2) developing SNP markers specific to known functional and fiber associated genes, (3) assigning EST-SSR and SNP markers to chromosomes, (4) linking EST-SSR and QTL for agronomic and fiber traits, and (5) linking molecular markers with genes for nematode resistance. Significant Activities that Support Special Target Populations 1)To expand cotton genetic diversity day-neutral selections were made in 68 F2 populations of exotic race stocks by a day neutral donor cultivar. Seed are being increased and agronomic evaluations will be conducted. The useful diversity in the day neutral lines can be used to improve commercial cotton cultivars. 2)Fifty photoperiodic exotic race lines were crossed to a day neutral donor parent. These new crosses will add needed diversity to cotton germplasm which will ultimately feed into cultivar development programs. 3)Broaden the genetic base for Upland cotton improvement. The narrow genetic base and limited information about genes controlling fiber and agronomic traits are two major impediments in the genetic improvement of Upland cotton. We reported on the development of three new interspecific backcrossed chromosome or chromosome arm substitution lines [CS-B 01, CS-B11sh (chromosome 11 short arm) and CS-B 12sh] and the association of agronomic and fiber traits with the alien chromosome or chromosome arm of these CS-B lines. We initiated the development of about 100 recombinant inbred lines (BC5S2) specific to each of three chromosomes (chromosome 25, 22sh and 22Lo).4) Develop and use PCR based DNA markers for improvement of upland cotton. There is a critical need to discover DNA markers associated with agronomic, fiber quality, and pest resistance and new sources of genetic diversity in Upland cotton to make U.S. in the forefront of competitive world cotton market. We surveyed genetic diversity of 620 wild race stocks and improved Upland cotton lines from Uzbekistan germplasm collection in two different environments and screened these lines with about 300 SSR markers to detect genetic diversity. We detected the existence of useful genetic diversity within this large germplasm collection and discovered several SSR markers associated with important fiber and agronomic traits that may be useful for marker assisted selection. We also detected genome wide linkage disequilibrium ranging from 30-50 cM and from 10-20 cM in exotic and improved Upland cotton genome, respectively, using association mapping strategy. This research will be useful to detect new source of genetic variation for the development of superior cotton germplasm. 5. Update on reniform nematode functional genomics. Approximately 2,000 reniform nematode expressed sequence tags (ESTs) were recovered from feeding sedentary females in an effort to identify nematode genes that may lend themselves to silencing through RNA-interference leading to plant resistance. Twenty ESTs have been found that show a high degree of similarity to Caenorhabiditis elegans genes that have a lethal phenotype following RNAi-mediated silencing. The remaining ESTs have been analyzed extensively using bioinformatic tools and the sequences will be deposited in GenBank once our analyses are complete. These data represent the first EST analysis of the reniform nematode and the corresponding data will be very useful to any laboratory studying renifom nematode. More experiments will determine the utility of the twenty candidate genes in providing RNAi-mediated plant resistance to reniform nemtode. NP301;Comp.3.
Impacts
(N/A)
Publications
- An, C., Saha, S., Jenkins, J.N., Scheffler, B.E., Wilkins, T.A., Stelly, D. M. 2007. Transcriptome profiling, sequence characterization, and SNP- based chromosomal assignment of the EXPANSIN genes in cotton. Molecular Genetics and Genomics. 278:539-553.
- Creech, R.G., Jenkins, J.N., McCarty Jr., J.C., Hayes, R.W., Creech, J.B., Haire, D., Cantrell, R. 2007. Registration of MS-01RKN, MS-24RKN, MS-30RKN, MS-33RKN, MS-35RKN and MS-37RKN cotton germplasm lines with resistance to root-knot nematode. Journal of Plant Registrations. 1:147-148.
- Hsu, C., An, C., Saha, S., Ma, D., Jenkins, J.N., Scheffler, B.E., Stelly, D. 2008. Molecular and SNP characterization of two genome specific transcription factor genes GhMyb8 and GhMyb10 in cotton species. Euphytica. 159:259-273.
- Guo, Y., Saha, S., Yu, J., Jenkins, J.N., Kohel, R.J., Scheffler, B.E., Stelly, D.M. 2008. BAC-derived SSR chromosome locations in cotton. Euphytica. 161:361-370.
- McCarty Jr., J.C., Wu, J., Jenkins, J.N. 2008. Genetic association of cotton yield with its component traits in derived primitive accessions crossed by elite Upland cultivars using the conditional ADAA genetic model. Euphytica. 161:337-352.
- An, C., Saha, S., Jenkins, J.N., Ma, D., Scheffler, B.E., Kohel, R.J., Yu, J., Stelly, D.M. 2008. Cotton (Gossypium spp.) R2R3-MYB transcription factors SNP identification, phylogenomic characterization, chromosome localization and linkage mapping. Theoretical and Applied Genetics. 161:1015-1026.
- Wubben, M., Callahan, F.E., Hayes, R.W., Jenkins, J.N. 2008. Molecular characterization and temporal expression analyses indicate that the MIC (Meloidogyne Induced Cotton) gene family represents a novel group of root- specific defense-related genes in upland cotton (Gossypium hirsutum L.). Planta. 228:111-123.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Broaden the genetic base for upland cotton improvement. Develop pest resistant germplasm, breeding methodologies and marker assisted selection. Identify genes and gene products correlated with resistance to nematodes. Develop and use PCR based DNA markers for improvement of upland cotton. Approach (from AD-416) Broaden the germplasm base of upland cotton and develop special breeding populations by (1) accessing genetic diversity in exotic photoperiodic accessions, (2) developing elite breeding populations through random mating, (3) developing backcrossed chromosome substitution lines, and (4) developing chromosome specific recombinant inbred lines for selected G. barbadense backcrossed chromosome substitution lines. Develop pest resistant germplasm, breeding methodologies, and agronomic and fiber evaluations by (1) evaluating for agronomic and fiber properties and genetic analyses, (2) evaluating lines for tobacco budworm tolerance or resistance, (3) evaluating industry developed transgenic insect resistant lines to provide a non-industry view on performance of specific transgenes and transformation events, (4) discovering and developing root- knot and reniform nematode resistant germplasm, (5) breeding methodologies for root-knot nematode resistance, and (6) conducting evaluations in a regional root-knot nematode nursery. Identify genes and gene products correlated with resistance to nematodes by (1) developing hairy root system in cotton, and (2) characterizing MIC genes. Develop and utilize PCR based DNA markers for improvement of upland cotton by (1) developing informative PCR based EST-SSR markers, (2) developing SNP markers specific to known functional and fiber associated genes, (3) assigning EST-SSR and SNP markers to chromosomes, (4) linking EST-SSR and QTL for agronomic and fiber traits, and (5) linking molecular markers with genes for nematode resistance. Significant Activities that Support Special Target Populations To expand cotton genetic diversity day-neutral selections were made in 92 F2 populations of exotic race stocks by a day neutral donor cultivar. Seed are being increased and agronomic evaluations will be conducted. The useful diversity in the day neutral lines can be used to improve commercial cotton cultivars. Fifty photoperiodic exotic race lines were crossed to a day neutral donor parent. These new crosses will add needed diversity to cotton germplasm which will ultimately feed into cultivar development programs. Putative reniform nematode resistance was identified in day neutral primitive cotton germplasm and individual plant selections were made following greenhouse tests. Seed from individual plant selections were increased, progeny were screened, and resistance was verified. Parental lines GB 713 and Nemx have been used to generate a segregating population. One marker has been found associated with resistance and additional markers are being screened against this population. Resistance when incorporated in cultivars will be valuable for cotton growers. Recombinant inbred lines (RIL) from crosses of various root-knot nematode resistant and susceptible parents have been developed. These are being used to genotype for nematode resistance and for molecular markers. Simple Satellite Repeat (SSR) markers associated with root-knot nematode resistance have been identified on chromosomes 11 and 14. MIC-3 characterization: An understanding of the molecular processes involved with nematode root interface during infection should provide information useful in understanding the genetics of resistance to nematodes. We identified fourteen cDNAs showing 86-99% identity with MIC- 3 that are expressed in M315 reniform nematode resistant (RNR) and/or M8. A time-course analysis of MIC-3 and selected defense gene expression in response to root-knot nematode infection was accomplished for M315 RNR and M8. Only MIC-3 showed increased expression in M315 RNR roots following infection (peroxidase, PR10, ERF5, and LOX1 did not show induction). Approximately 30 potential transgenic hairy root lines were developed in an effort to over-express the MIC-3 genomic open reading frame in a nematode-susceptible genetic background, i.e., DP90. Analysis of these putative transgenic hairy root lines is ongoing. Accomplishments Root-knot nematode resistant cotton germplasm released. Root-knot nematode is a major pest in cotton and resistant cultivars would provide an ideal solution, but breeders need germplasm coupling resistance genes with genes for good agronomic and fiber quality. We developed and released six root-knot resistant germplasm lines. These lines provide germplasm with high levels of resistance to root-knot nematode plus good agronomic and fiber properties. Most major cotton seed breeding companies requested and received seed of these lines for use in their breeding program. NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Crops; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Random mated population of cotton released. The germplasm used by major breeding companies is more narrow than desired and this limits breeding progress for new cultivars with improved fiber quality and yield. We developed and released a population after 6 cycles of random mating beginning with a diverse 11 parent diallele. All major cottonseed breeding companies have requested and received seed of this population. This provides companies with a broad based germplasm that has reshuffled the genes for fiber quality and yield. This is very valuable germplasm for broadening the germplasm base from which varieties are developed. NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Cotton; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Day Neutral lines derived from primitive cotton accessions are useful for cotton improvement. The germplasm used by major breeding companies is more narrow than desired and this limits breeding progress for new cultivars with improved fiber quality and yield. One-hundred fourteen day- neutral derived germplasm lines from race accessions collected from nine regions, covering 16 countries were evaluated in field trials for two years. These derived germplasm lines provide genes with significant additive effect for fiber qualities, while the additive effects for yield improvement were not significantly decreased. The genetic resources from the accessions showed no consistent effect of collection location or geographic race. These derived lines offer the potential to improve important traits and expand genetic diversity. NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Cotton; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. There is a critical need of DNA markers that provide specific information as to their role in biological activities in cotton. Recently, single- nucleotide polymorphisms (SNPs) have become an efficient tool in genome mapping. We cloned, sequenced, and characterized several members of six EXPANSIN A and eight Myb genes associated with fiber development. We discovered a strategy of identifying SNP markers associated with these genes in tetraploid cotton and localized the markers to chromosomes. These markers will be useful to tag candidate genes with complex fiber and agronomic traits in cotton molecular mapping programs. DNA sequences were submitted to GenBank of National Center for Biotechnology Information (NCBI). NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Cotton; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Broadening cotton germplasm base: The lack of progress in improving Upland cotton yield and fiber properties suggests that these traits could be improved by introgressing genes from outside the primary germplasm pool. One approach to introgressing genes into an Upland background from other species would be to use interspecific chromosome substitution lines. We discovered chromosomal association of many agronomic and fiber traits using intercrosses of CS-B lines (chromosome substitution lines from G. barbadense) and revealed that cryptic beneficial alleles for fiber yield and qualities exist in the unadapted CS-B lines that can be useful in the genetic improvement of Upland cotton. NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Cotton; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Cotton hairy roots: A method of conducting research in the laboratory with nematodes and roots is needed. Non-transgenic hairy root cultures corresponding to M315 RNR, Nemex, and GB-713 (reniform nematode resistant) were developed. A reproducible method for inoculating cotton hairy roots with root-knot and reniform nematode was also developed. We determined that M315 RNR hairy roots showed resistance to root-knot nematode and that GB-713 hairy roots showed resistance to reniform nematode compared to a nematode-susceptible hairy root culture (DP90). NP 301: Plant Genetic Resources, Genomics, and Genetics Improvement; Component 3: Genetic Improvement of Cotton; Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Technology Transfer Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 6
Impacts
(N/A)
Publications
- Ynturi, P., Jenkins, J.N., McCarty Jr., J.C., Gutierrez, O.A., Saha, S. 2006. Association of root-knot nematode resistance genes with simple sequence repeat markers on two chromosomes in cotton. Crop Science. 46:2670-2674.
- Saha, S., Raska, D.A., Stelly, D.M. 2006. Upland (Gossypium hirsutum L.) x Hawaiian cotton (G. tomentosum Nutt. ex Seem.) F1 hybrid hypoaneuploid chromosome substitution series. Journal of Cotton Science. 10:263-272.
- Gutierrez, O.A., Bowman, D.T., Cole, C.B., Jenkins, J.N., McCarty Jr., J.C. , Wu, J., Watson, C.E. 2006. Development of random-mated populations using bulked pollen methodology: Cotton as a model. Journal of Cotton Science. 10:175-179.
- McCarty Jr., J.C., Wu, J., Saha, S., Jenkins, J.N., Hayes, R.W. 2006. Effects of chromosome 5sh from Gossypium hirsutum L. on flower production in G. hirsutum L. Euphytica. 152:99-107.
- McCarty Jr., J.C., Wu, J., Jenkins, J.N. 2007. Use of primitive derived cotton accessions for agronomic and fiber traits improvement: Variance components and genetic effects. Crop Science. 47:100-110.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C., Zhong, M., Swindle, M. 2007. AFLP marker associations with agronomic and fiber traits in cotton. Euphytica. 153:153-163.
- Jenkins, J.N., McCarty Jr., J.C., Wu, J., Saha, S., Gutierrez, O.A., Hayes, R.W., Stelly, D.M. 2007. Genetic effects of thirteen Gossypium barbadense L. chromosome substitution lines in topcrosses with upland cotton cultivars: II. Fiber quality traits. Crop Science. 47:561-572.
- Abdurakhmonov, I.Y., Kushanov, F., Djaniqulov, F., Buriev, Z., Pepper, A.E. , Fayzieva, N., Mavlonov, G., Saha, S., Jenkins, J.N., Abdukarimov, A. 2007. The role of induced mutation in conversion of photoperiod dependence in cotton. Journal of Heredity. 98:258-266.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This research project is assigned to National Program (NP) 301 and the component, Genetic characterization, manipulation, and genetic improvement of cotton. It also contributes solutions to National Program 302 and the component, Plant defense and secondary metabolism, and National Program 304 and the component, Development of new and improved pest control technologies and integration of component technologies for IPM systems. Objective 1: Broaden the genetic base for upland cotton improvement. Objective 2: Develop pest resistant germplasm, breeding methodologies and marker assisted selection. Objective 3: Identify genes and gene products associated with resistance to nematodes. Objective 4: Develop and use polymerase chain reaction (PCR) based DNA markers for improvement of upland
cotton. The approaches are: Approach 1: Broaden the germplasm base of upland cotton and develop special breeding populations by (a) accessing genetic diversity in exotic photoperiodic accessions; (b) developing elite breeding populations through random mating; (c) developing backcrossed chromosome substitution lines; and (d) developing chromosome specific recombinant inbred lines for selected chromosome substitution lines. Approach 2: Develop pest resistant germplasm, breeding methodologies, and agronomic and fiber evaluations by (a) evaluating lines for agronomic and fiber properties and genetic analyses; (b) evaluating lines for tobacco budworm resistance; (c) evaluating industry developed transgenic insect resistant lines for performance of specific transgenes and transformation events; (d) discovery and development of root-knot and reniform nematode resistant germplasm; (e) development of breeding methodologies for root-knot nematode (RKN) resistance; and (f) conduct
evaluations of breeding lines in a regional RKN nursery. Approach 3: Identify genes and gene products correlated with resistance to nematodes by (a) developing hairy root system in cotton and (b) characterizing Meloidogyne-induced cotton (MIC) genes. Approach 4: Development and utilization of PCR based DNA markers for improvement of upland cotton by (a) developing informative PCR based Express Sequence Tag (EST)-Simple Sequence Repeat (SSR) markers, (b) developing single nucleotide polymorphisms (SNP) markers specific to known functional and fiber associated genes, (c) assigning EST-SSR and SNP markers to chromosomes, (d) linking EST-SSR and quantitative trait loci (QTL) for agronomic and fiber traits, and (e) linking molecular markers with genes for nematode resistance. Cotton growers are experiencing shrinking profits in cotton production. This is due largely to higher input costs without increases in yield or quality; thus, many producers are having a difficult time continuing
to produce cotton profitably. Genetic diversity among varieties and breeding programs needs to be expanded because breeders depend upon this genetic diversity to breed new and improved varieties. Research shows that all current varieties are closely related. Tobacco budworm, cotton bollworm, and nematodes are the most serious insect pests of cotton in the U.S. Control costs for insects range from $60 to $90 per acre. Insects are resistant to many insecticides. There are no commercial varieties resistant to reniform nematode and none with a high level of resistance to RKN; however, the level of RKN resistance is increasing in commercial cultivars. Our research program has discovered and developed germplasm with a very high level of resistance to RKN; however, there is not a good selection method for use in applied breeding. Molecular markers linked with nematode resistance should make breeding resistant varieties feasible. Very little is known about the genomic location of important
genes for agronomic, fiber, and pest resistance. Knowledge is limited about the molecular biology of cotton. This area needs expanded research. This is a critical time in cotton production, as costs are escalating, pests are developing increased levels of resistance to pesticides, nematode pests are increasing, and prices growers receive continue to be low. The genetic base that cotton breeders use in applied breeding programs is narrow. The genetic base in the genus Gossypium is large, but greatly limited in its usefulness to applied breeders because of photoperiodicity in exotic G. hirsutum accessions, cross incompatibility problems associated with interspecific crosses, and the lack of molecular knowledge and/or markers in cotton that allows breeders to combine conventional and molecular marker approaches in breeding. Thus, diversity exists in the genus, but requires discovery, early stage breeding, linkage of DNA markers with QTL and pest resistance genes in order to make the
needed advances in development of new varieties with enhanced pest resistance, improved fiber, and increased yield. DNA markers linked to agronomic traits should significantly increase the efficiency of various breeding strategies via marker assisted selection. The two major limiting factors in the use of molecular markers in cotton are: 1) limited number of markers available in the public sector and 2) lack of marker association with economically important QTL in cotton. The tetraploid species, G. barbadense, G. tomentosum, and G. mustelinum, are reservoirs of important genes for pest and disease resistance, and for improved agronomic and fiber traits. Breeders face many challenges when introgressing genes from these species. An alternative approach to conventional introgression of alien genes for improved traits into an Upland background is via chromosome substitution lines. New transgenes and new natural genes are needed for pest resistance, and scientists in this research project
will seek to discover new genes for pest resistance. Nematode pests reduce profits for cotton growers. The National Cotton Council estimated a loss in yield equivalent to about $250 million per year due to nematode pathogenesis. Profits are lost through money spent for conventional pesticide control and through yield losses from failure to completely control nematode populations. Plant genes for resistance to these pests should offer an effective control approach. Genes for nematode resistance exist in Gossypium, but breeding improved cotton varieties with nematode resistance is difficult due to labor intensive, time consuming bioassays required to identify resistant progeny. Molecular approaches offer a way to improve the speed of development in breeding programs through marker assisted selection and identification of resistance genes for genetic engineering. To date no genes for RKN or reniform nematode resistance in cotton have been isolated and sequenced. The work proposed here
characterizing gene expression during early interaction of roots with nematode should provide useful tools to aid the incorporation and use of resistance genes for control of nematodes in cotton. New transgenes for pest resistance are being developed by several commercial companies. As industry develops these transgenes we will cooperate with companies that ask us to do so. We presently have trust agreements with three companies. 2. List by year the currently approved milestones (indicators of research progress) FY 2004: Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of chromosome substitution lines of G. barbadense in G. hirsutum. FY 2005: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Develop hairy root system in cotton for nematode research. Make
germplasm release of RKN resistant lines with improved agronomic traits. Develop new linkage map of SSR and QTL in recombinant inbred lines (RIL) from an intraspecific cross. Report and deposit in public database 100 EST containing SSR markers. Complete development of RIL for 4 populations from crosses of nematode resistant x susceptible lines. FY 2006 Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of random mated populations involving 4 cycles of random mating with 11 elite lines. Make germplasm release of additional chromosome substitution lines. Report on discovery of genes for resistance to reniform nematode. Add about 100 Brookhaven National Laboratory (BNL) SSR to intraspecific RIL linkage map. FY 2007 Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Complete development of 3
chromosome specific RIL from chromosome substitution lines. FY 2008: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Complete development of chromosome substitution lines from G. tomentosum in G. hirsutum. Identify PCR based markers closely associated with RKN resistance. Report characterization of MIC gene in RKN resistant lines. Complete linkage map of several kinds of PCR based markers including SNPs. 4a List the single most significant research accomplishment during FY 2006. Chromosomes Identified for RKN Resistance Genes in Cotton: The two major genes in cotton responsible for resistance to RKN were discovered to be located on chromosomes 11 and 14 by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Mississippi State University. This addresses the need that commercial plant breeders have relative to the number of major genes involved in
RKN resistance. Breeding for RKN resistance is still complicated by the need for better technology for identifying individual resistant plants in segregating populations used in commercial plant breeding. We confirmed our earlier work showing that two major genes were involved and have now located these to chromosome and have made substantial progress on DNA markers associated with these genes. This information should aid applied breeders in developing RKN resistant cultivars. 4b List other significant research accomplishment(s), if any. RKN Germplasm Developed: RKN resistant germplasm with good agronomic and fiber traits was developed by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Mississippi State University and Cotton Incorporated. This germplasm provides genes for RKN resistance in improved lines and will be useful for breeding cultivars with improved agronomic and fiber traits combined with RKN resistance. The Auburn 634
source of RKN resistance was combined with good agronomic and fiber quality genes by crossing with cultivars followed by selection. This germplasm is being released and should speed the development of RKN resistant cultivars for growers. Improving Cotton Cultivars with Genes from G. barbadense: The utility of chromosome substitution (CS-B) lines of cotton as parents to introgress genes on specific G. barbadense chromosomes into specific cultivars of upland cotton was determined in cooperative research between the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Texas A&M University. This addresses the need for information relative to how the chromosome substitution lines can be used in applied breeding programs and bridges the gap between basic genetics and applied plant breeding. Five diverse cultivars representing the major cotton breeding companies were crossed with 13 CS-B lines and parents and F2 generations were grown in 4 environments. This
provided commercial breeders with specific information about the utility of using these 13 specific CS-B lines with their cultivars and provides them with the necessary information to assist in making informed choices of parents to use in crosses to develop cultivars with genes introgressed from G. barbadense. Germplasm Registrations from Primitive Accessions: Germplasm registrations involving 35 improved day neutral germplasm lines from primitive accessions were made by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This addresses the need to increase diversity of cotton breeding lines. These germplasm lines were previously released and have now been registered with Crop Science Society of America. These are from crosses of photoperiodic accessions with cultivars followed by selection of day neutral plants and evaluation in field plots. Seed of these germplasms are now inventoried in the National Germplasm System and are available to geneticists and
breeders. Their presence in the National Germplasm System should make knowledge of them widely known to breeders around the world. Genes Useful in Cotton Breeding Discovered in Exotic Accessions: A group of 114 day neutral lines from exotic accessions were developed and evaluated in two environments by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This germplasm addresses the need for new sources of useful genes for fiber quality for breeders to use in developing improved cultivars. The germplasm lines had improved fiber length, strength, micronaire, and F2 yields comparable to cultivars when crossed with two widely adapted cultivars; however, lint percentage was lower. These day neutral lines provide genes to improve important traits and expand genetic diversity for cotton breeders and will help in developing commercial cultivars with improved fiber quality. New Genetic Models and Software for Analysis of Genetics and Plant Breeding Experiments: Four
manuscripts describing genetic models, software, and data analysis for plant breeding experiments were published through cooperative research between the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Mississippi State University. These address the need for better statistical models to partition, understand, and utilize genetic variances in plant breeding. These methodologies describe a recursive approach to detect multivariable conditional variance components, compare two statistical models for evaluating boll retention in cotton, provide variance component estimation using the additive, dominance, and additive by additive model when genotypes vary across environments, and an additive dominance model to determine chromosomal effects in chromosome substitution lines and other germplasm. These software, models, and data set analyses provide new and advanced techniques for plant breeders that should improve their ability to obtain maximum information from
their data sets which should in turn improve their plant breeding efforts. CS-B Lines Registered: Germplasm registration of 17 chromosome substitution germplam lines were made by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Texas A&M University. This addresses the need to increase diversity of cotton breeding lines and new technologies to introgress genes from G. barbadense into upland cotton. These germplasms were previously released and have now been registered with Crop Science Society of America. Their presence in the National Germplasm System should make knowledge of them widely known to breeders around the world. 4d Progress report. Resistance to Reniform Nematode Discovered in Exotic Cotton Accessions: Putative reniform nematode resistance was identified in day neutral primitive cotton germplasm developed by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This addresses a serious cotton production problem as
all cultivars available to growers are susceptible to reniform nematode and breeders do not have a good source of genes for resistance. Seed from individual plant selections have been increased and will be evaluated in greenhouse tests to determine level of resistance to reniform nematode. Since no commercial cultivars carry resistance to reniform nematode this resistant source will be valuable to cotton breeders to use as a resistant parent for developing resistant cultivars for cotton growers. Inheritance of Resistance to Reniform Nematode: Inheritance of resistance to reniform nematode from a Gossypium barbadense source, GB- 713, was determined through cooperative research between the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and ARS at College Station, TX. This addresses the need for a source of resistance genes and information of inheritance of genes for resistance to reniform nematode. Parental lines, GB-713 and NemX, were crossed and 4
segregating generations were inoculated with reniform nematode, and reproduction was determined. Inheritance was determined to be due to a single partially dominant gene and we are currently determining association of this resistance gene with SSR markers. Breeders can immediately begin using accession GB-713 as a source of a resistance gene to develop cultivars resistant to reniform nematode. Random Mating Population Developed for Cotton Breeding: A random mated population was developed through five cycles of random mating beginning with a diallele cross among 11 elite lines by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, cooperating with Mississippi State University, North Carolina State University, and Cotton Incorporated. This addresses the breeding problem of adverse linkages between fiber quality genes and yield genes that complicate breeding efforts for better fiber quality and increases in yield. The first crosses were made as an 11 parent
diallele using diverse parents from all areas of the U.S. cotton belt. Five cycles of random mating have now been completed. Fingerprinting of the 11 parental lines continues with SSR markers. Germplasm release of these random mating populations are planned and will provide valuable diversity and new gene combinations useful to all public and private cotton breeding programs. Genetic Diversity for Cotton Breeding: Day-neutral selections were made in 98 F2 populations of exotic race stocks crossed with a day neutral donor cultivar by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This addresses the need to expand the genetic base of parental lines used in breeding improved cultivars. Crosses were made, followed by selection, and seed were increased for agronomic evaluations. Genes in the day neutral lines can be used to improve commercial cotton cultivars. Genes for Increased Flower Number Discovered: Thirteen chromosome substitution lines were
evaluated by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, for number of flowers produced, and CS-B05sh produced more flowers than its donor parent 3-79 or its recurrent parent TM1. Cotton yield is primarily determined by the number of mature bolls that develop from squares and flowers, and this research addresses the problem of new genes for yield improvement. This illustrated how the chromosome substitution lines are a new technology to move genes from G. barbadense into upland cultivars in breeding programs. Basic understanding of flower production may guide breeders in ways to increase yield in cotton. New CS-B Lines Developed: Four new CS-B lines were developed and these 4 plus 5 previously developed lines were evaluated in crosses with TM-1 for agronomic and fiber traits by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Texas A&M University. This addresses the need for better ways to introgress
genes from G. barbadense into upland cotton. We discovered chromosomal association of important fiber and agronomic traits in these germplasms. We have previously developed and made available 17 CS-B lines. Together these represent unique germplasm useful for introgression of genes from G. barbadense into upland cotton in practical breeding programs. These CS-B lines provide a unique and useful way to improve agronomic and fiber properties in applied cotton breeding. New Transgenes for Insect Resistance Evaluated: Four experiments evaluating transgenic events and combination of events are being evaluated for efficacy of transgenes to control cotton bollworm by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with industry. This addresses the need for additional insect transgenes by growers and the need for better ways to implement resistance management. Plots are being grown with natural or artificial infestations of cotton bollworm. When
available to growers in cultivars with new transgenes for insect resistance this will provide more choices to growers as well as aid in insect resistance management. Linkage of SSR Markers with Lint Percentage in Cotton: There were 12 SSR markers linked with lint percentage discovered in a set of RIL G. hirsutum by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with The Laboratory of Genetic Engineering and Biotechnology, Institute of Genetic and Plant Experimental Biology, Academy of Sciences of Uzbekistan; and Texas A&M University. This addresses the need for markers associated with lint traits in cotton. These 12 markers were located on chromosomes 12, 18, 23, and 26. Some of the markers were from bacterial artificial chromosomes; thus, these may be useful for map based cloning of lint percentage genes in cotton. A better understanding of the genes involved in fiber production in cotton could result from this research. SSR Markers
Associated with Day Neutrality Traits in Cotton: SSR markers for the genes responsible for day neutral traits in radiation induced mutant cotton lines developed in Uzbekistan were discovered by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperative research with The Laboratory of Genetic Engineering and Biotechnology, Institute of Genetic and Plant Experimental Biology, Academy of Sciences of Uzbekistan; and Texas A&M University. This addresses the need for a better understanding of flowering in cotton. These markers should help us isolate and understand the genetics of day neutrality in cotton and to eventually more easily use the exotic accessions of photoperiodic cotton. SNP Markers Specific to MIC-3, a Root Specific Gene in Cotton: Five family members of the MIC-3 root specific gene family were identified and found to be clustered on the short arm of chromosome 4, in cooperative research among the Genetics and Precision Agriculture Research
Unit at Mississippi State, MS; The Laboratory of Genetic Engineering and Biotechnology, Institute of Genetic and Plant Experimental Biology, Academy of Sciences of Uzbekistan; and Texas A&M University. This addresses the problem of the need for new types of genetic markers in cotton research. These gene sequences had high homology with EST sequences in GeneBank that were isolated from cotton plants infected with RKN and Fusarium Wilt. SNPs were identified among the family members. This research provides a strategy for discovery of putative SNP markers associated with candidate pest resistance genes in tetraploid cotton. SSR Linkage Map with Chromosomal Assignment of Markers: The most complete G. hirsutum SSR linkage map to date is an SSR linkage map constructed using a recombinant inbred population from the cross of two G. hirsutum lines by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and scientists at Texas A&M University. This addresses the need
for a G. hirsutum based linkage map useful for plant breeding. Markers were assigned to linkage groups and linkage groups confirmed to chromosome using known cytogenetic stocks. This linkage map provides cotton geneticists with the best marker information available for direct use in cotton breeding. Cotton Microsatellite Database (CMD) Developed: A public database of cotton microsatellite markers has been developed through the cooperation of ARS, Clemson University, CIRAD, Texas A&M University, Brookhaven National Lab, Monsanto, Nanjing Agricultural University, Delta and Pine Land Seed Company, Bayer BioScience, Cotton Incorporated, and Washington State University. This addresses the need for more markers to be deposited in a public database for cotton. The CMD is available on the web at http://www.cottonssr.org. This database provides researchers with a centralized access to microsatellite markers which are an invaluable resource for basic and applied cotton genetics and breeding
research. 5. Describe the major accomplishments to date and their predicted or actual impact. New Germplasm Released for use in Cotton Breeding: The Genetics and Precision Agriculture Research Unit at Mississippi State, MS, made germplasm releases of several high strength fiber lines in FY 2004 which can be used by industry to breed improved fiber quality cultivars. Thirty- five day neutral lines derived from exotic G. hirsutum photoperiodic germplasm have been released as germplasm for use in cultivar development. A group of 114 day neutral lines from exotic accessions were developed and evaluated in two environments. These germplasm lines have improved fiber length, strength, micronaire, and F2 yields comparable to cultivars when crossed with two widely adapted cultivars; however, lint percentage was lower. These day-neutral lines provide genes to improve important traits and expand genetic diversity for cotton breeders and will help in developing commercial cultivars with improved
fiber quality. A germplasm release was made of 17 chromosome substitution lines in which chromosomes from G. barbadense have been substituted into G. hirsutum lines. In addition, in 2006, we published a manuscript that provides very useful information to commercial breeders on the genetic interactions between the chromosome substitution lines and cultivars from specific commercial breeding programs. This information should greatly aid in the use and utility of the chromosome substitution lines. Resistance to Reniform Nematode in G. hirsutum Exotic Accessions: Scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Agricultural Research Service (ARS) at College Station, TX, identified resistance to reniform nematode in two wild accessions which we had converted to day neutrality. Reniform nematode is a serious pest for growers of upland cotton and no good plant resistance is available in cultivars. This germplasm provides genes for plant
breeders to use to develop resistant cotton cultivars which should provide an economic and environmentally friendly method for growers to use to control this destructive pest. Chromosomes Identified for RKN Resistance Genes in Cotton: The two major genes in cotton responsible for resistance to RKN were discovered to be located on chromosomes 11 and 14 by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Mississippi State University. This addresses the need that commercial plant breeders have relative to the number of major genes involved in RKN resistance. We confirmed that two major genes were involved in resistance and located these to chromosome and have made substantial progress on DNA markers associated with these genes. This information should aid applied breeders in developing RKN resistant cultivars. New Statistical Models and Software for Analyzing Plant Breeding Experiments: The Genetics and Precision Agriculture Research
Unit at Mississippi State, MS, in cooperation with Mississippi State University have developed and published several new statistical analyses and methodologies for analyzing plant breeding experiments. These provide new approaches to obtaining useful information from plant breeding experiments in basic and applied research. Plant breeders can use these models to analyze their data and obtain a better understanding of the genetics of their breeding lines and should result in better plant selections and thus speed up the development of improved cultivars for grower use. Molecular Markers Added to Cotton: The Genetics and Precision Agriculture Research Unit in cooperation with Alabama A&M University identified 133 SSR-containing consensus EST sequences. This is the first report on PCR-based EST-SSR markers in cotton. More than 50% of these EST- SSRs are associated with fiber ESTs. In cooperation with ARS scientists at Stoneville, MS, and College Station, TX, and Texas A&M University
scientists we determined the chromosomal association of 50 bacterial artificial chromosome (BAC) derived SSR loci. Chromosome based BAC- derived SSR markers will provide bridges that will help integrate physical maps with genetic maps and will provide a useful framework of markers for chromosome based genome sequencing and facilitate positional candidate gene cloning, comparative genome analysis, and a platform in the future for chromosome based genome sequencing projects. These discoveries aid both basic and applied cotton genomics research scientists. Chromosomal Location of a Cyclin-dependent Kinase Fiber Gene: A SNP associated with a cyclin dependent kinase A gene was located to the long arm of chromosome 16 through cooperative research among scientists in the Geneticis and Precision Agriculture Research Unit at Mississippi State, MS, Mississippi State University, and Texas A&M University. With this discovery we demonstrated that single primer extension technology could be used to
identify SNP markers associated with cloned and sequenced fiber genes in cotton. This addresses the problem of the need for a better understanding of the genetics of fiber development in cotton. As these and additional fiber genes are located to chromosomes, plant breeders will have a better understanding of how to manipulate genes for fiber quality in cultivar development. New Assignment of SSR Markers to Chromosomes: From a screen of 397 BNL SSR markers, 142 were assigned to specific chromosomes, linkage group A02 was assigned to chromosomes 8, A03 was assigned to chromosome 11, and SSR markers were assigned to chromosome by using interspecific hyperaneuploid hybrids of G. hirsutum x G. tomentosum and G. hirsutum x G. barbadense. This was accomplished by cooperative research between scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and scientists at Texas A&M University. This addresses the problem of a need for assignment of framework
markers to all the chromosomes in cotton. These new and confirmed marker assignments will greatly accelerate the construction of useful linkage maps and the location of QTL linked to fiber, agronomic, and pest resistance genes in upland cotton. Twenty-four manuscripts were published in FY 2004, 23 in FY 2005, and 11 in FY 2006 on research in this project. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Several germplasm lines with genes for needed traits to improve cotton cultivars have been developed and released. These include genes for improved fiber quality and nematode resistance. These are immediately available for use by the first set of end users which are public scientists and scientists in commercial breeding firms. As they use these
germplasms to improve cultivars, these genes in the form of improved cultivars, will become available to the final end users which are the growers. Manuscripts have been published describing new statistical methodologies for genetic analyses of plant breeding experiments. These can be immediately used by all plant breeders. These methodologies should improve the plant breeders ability to identify and utilize genes in their breeding lines.
Impacts
(N/A)
Publications
- McCarty Jr., J.C., Jenkins, J.N. 2005. Registration of 21 day length- neutral flowering primitive cotton germplasm lines. Crop Science. 45:2134.
- Wu, J., Wu, D., Jenkins, J.N., McCarty Jr., J.C. 2006. A recursive approach to detect multivariable conditional variance components and conditional random effects. Computational Statistics and Data Analysis. 50:285-300.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C., Watson, C.E. 2005. Comparisons of two statistical models for evaluating boll retention in cotton. Agronomy Journal. 97:1291-1294.
- McCarty Jr., J.C., Jenkins, J.N. 2005. Registration of 14 primitive derived cotton germplasm lines with improved fiber strength. Crop Science. 45:2668.
- Stelly, D.M., Saha, S., Raska, D.A., Jenkins, J.N., McCarty Jr., J.C., Gutierrez, O.A. 2005. Registration of 17 upland (Gossypium hirsutum) cotton germplasm lines disomic for different G. barbadense chromosome or arm substitutions. Crop Science. 45:2663-2665.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C., Wu, D. 2006. Variance component estimation using the additive, dominance, and additive x additive model when genotypes vary across environments. Crop Science. 46:174-179.
- Saha, S., Jenkins, J.N., Wu, J., McCarty Jr., J.C., Gutierrez, O.A., Percy, R.G., Cantrell, R.G., Stelly, D.M. 2006. Effects of chromosome-specific introgression in upland cotton on fiber and agronomic traits. Genetics. 172:1927-1938.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C., Saha, S., Stelly, D.M. 2006. An additive-dominance model to determine chromosomal effects in chromosome substitution lines and other germplasms. Theoretical and Applied Genetics. 112:391-399.
- Jenkins, J.N., Wu, J., McCarty Jr., J.C., Saha, S., Gutierrez, O.A., Hayes, R.W., Stelly, D.M. 2006. Genetic effects of thirteen Gossypium hirsutum L. chromosome substitution lines in topcrosses with upland cotton cultivars: I. Yield and yield components. Crop Science. 46:1169-1178.
- McCarty, J.C., Wu, J., Jenkins, J.N. 2006. Genetic diversity for agronomic and fiber traits in day-neutral primitive cotton germplasm. Euphytica. 148:283-293.
- Gutierrez, O.A., Wu, J., Jenkins, J.N., McCarty, Jr., J.C., Raska, D.A., Stelly, D.M. 2006. An intraspecific SSR linkage map of cotton. National Cotton Council Beltwide Cotton Conference. p. 826.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This research project is assigned to National Program 301 and the component, Genetic Characterization, Manipulation, and Genetic Improvement of Cotton. It also contributes solutions to National Program 302 and the component, Plant Defense and secondary Metabolism and National Program 304 and the component, Development of New and Improved Pest Control Technologies and Integration of Component Technologies for IPM Systems. Objective 1: Broaden the genetic base for upland cotton improvement. Objective 2: Develop pest resistant germplasm, breeding methodologies, and marker assisted selection. Objective 3: Identify genes and gene products associated with resistance to nematodes. Objective 4: Develop and use PCR based DNA markers for improvement of upland cotton. The approaches are: Approach 1:
Broaden the germplasm base of upland cotton and develop special breeding populations by (a) accessing genetic diversity in exotic photoperiodic accessions, (b) developing elite breeding populations through random mating, (c) developing backcrossed chromosome substitution lines, and (d) developing chromosome specific recombinant inbred lines for selected chromosome substitution lines. Approach 2: Develop pest resistant germplasm, breeding methodologies, and agronomic and fiber evaluations by, (a) evaluating lines for agronomic and fiber properties and genetic analyses, (b) evaluating lines for tobacco budworm resistance, (c) evaluating industry developed transgenic insect resistant lines for performance of specific transgenes and transformation events, (d) discovery and development of root-knot and reniform nematode resistant germplasm, (e) development of breeding methodologies for root-knot nematode resistance, and (f) conduct evaluations of breeding lines in a regional root-knot
nematode nursery. Approach 3: Identify genes and gene products correlated with resistance to nematodes by (a) developing hairy root system in cotton and (b) characterizing MIC genes. Approach 4: Development and utilization of PCR based DNA markers for improvement of upland cotton by (a) developing informative PCR based EST-SSR markers, (b) developing SNP markers specific to known functional and fiber associated genes, (c) assigning EST-SSR and SNP markers to chromosomes, (d) linking EST-SSR and QTL for agronomic and fiber traits, and (e) linking molecular markers with genes for nematode resistance. Cotton growers are experiencing shrinking profits in cotton production. This is due largely to higher input costs without increases in yield or quality; thus, many producers are having a difficult time continuing to produce cotton profitably. Genetic diversity among varieties and breeding programs needs to be expanded because breeders depend upon this genetic diversity to breed new and
improved varieties. Research shows that all current varieties are closely related. Tobacco budworm, cotton bollworm, and nematodes are the most serious pests of cotton in the US. Control costs for insects range from $60 to $90 per acre. Insects are resistant to many insecticides. There are no commercial varieties resistant to reniform nematode and none with a high level of resistance to root-knot nematode; however, the level of root-knot nematode resistance is increasing in commercial cultivars. Our research program has discovered and developed germplasm with a very high level of resistance to root-knot nematode; however, there is not a good selection method for use in applied breeding. Molecular markers linked with nematode resistance should make breeding resistant varieties feasible. Very little is known about the genomic location of important genes for agronomic, fiber, and pest resistance. Knowledge is limited about the molecular biology of cotton. This area needs expanded
research. This is a critical time in cotton production, as costs are escalating, pests are developing increased levels of resistance to pesticides, nematode pests are increasing, and prices growers receive continue to be low. The genetic base that cotton breeders use in applied breeding programs is narrow. The genetic base in the genus Gossypium is large, but greatly limited in its usefulness to applied breeders because of photoperiodicity in exotic G. hirsutum accessions, cross incompatibility problems associated with interspecific crosses, and the lack of molecular knowledge and/or markers in cotton that allows breeders to combine conventional and molecular marker approaches in breeding. Thus, diversity exists in the genus, but requires discovery, early stage breeding, and linkage of DNA markers with QTL and pest resistance genes, in order to make the needed advances in development of new varieties with enhanced pest resistance, improved fiber, and increased yield. DNA markers
linked to agronomic traits should significantly increase the efficiency of various breeding strategies via marker assisted selection. The two major limiting factors in the use of molecular markers in cotton are 1) limited number of markers available in the public sector and 2) lack of marker association with economically important QTL in cotton. The tetraploid species, G. barbadense, G. tomentosum, and G. mustelinum, are reservoirs of important genes for pest and disease resistance, and for improved agronomic and fiber traits. Breeders face many challenges when introgressing genes from these species. An alternative approach to conventional introgression of alien genes for improved traits into an Upland background is via chromosome substitution lines. New transgenes and new natural genes are needed for pest resistance, and scientists in this research project will seek to discover new genes for pest resistance. Nematode pests reduce profits for cotton growers. The National Cotton
Council estimated a loss in yield equivalent to about $250 million per year due to nematode pathogenesis. Profits are lost through money spent for conventional pesticide control and through yield losses from failure to completely control nematode populations. Plant genes for resistance to these pests should offer an effective control approach. Genes for nematode resistance exist in Gossypium, but breeding improved cotton varieties with nematode resistance is difficult due to labor intensive, time consuming bioassays required to identify resistant progeny. Molecular approaches offer a way to improve the speed of development in breeding programs through marker assisted selection and identification of resistance genes for genetic engineering. To date no genes for root-knot nematode (RKN) or reniform nematode resistance in cotton have been isolated and sequenced. The work proposed here characterizing gene expression during early interaction of roots with nematode should provide useful
tools to aid the incorporation and use of resistance genes for control of nematodes in cotton. New transgenes for pest resistance are being developed by several commercial companies. As industry develops these transgenes, when asked we will cooperate with companies. We presently have trust agreements with three companies. 2. List the milestones (indicators of progress) from your Project Plan. FY 2004: Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of chromosome substitution lines of G. barbadense in G. hirsutum. FY 2005: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Develop hairy root system in cotton for nematode research. Make germplasm release of root-knot nematode resistant lines with improved agronomic traits. Develop new linkage map of SSR
and QTL in RIL lines from an intraspecific cross. Report and deposit in public database 100 EST containing SSR markers. Complete development of RIL for 4 populations from crosses of nematode resistant x susceptible lines. FY 2006: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of random mated populations involving 4 cycles of random mating with 11 elite lines. Make germplasm release of additional chromosome substitution lines. Report on discovery of genes for resistance to reniform nematode. Add about 100 BNL SSR to intraspecific RIL linkage map. FY 2007: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Complete development of 3 chromosome specific RIL from chromosome substitution lines. FY 2008: Develop about 20 day neutral race accession lines. Evaluate new transgenic constructs
from industry for resistance to Lepidoptera insects. Complete development of chromosome substitution lines from G. tomentosum in G. hirsutum. Identify PCR based markers closely associated with root-knot nematode resistance. Report characterization of MIC gene in root-knot nematode resistant lines. Complete linkage map of several kinds of PCR based markers including SNPs. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Develop about 20 day neutral race accession lines. Registered 35 day neutral germplasms with Crop Science. Milestone Fully Met 2. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Evaluated 14 lines from Bayer Crop Science and 4 lines from Dow AgroSciences. Milestone Fully Met 3. Develop hairy root system in cotton for nematode research. Obtained a hairy root system from USDA, ARS, Cotton Structure and
Quality Research Unit (CSQRU), New Orleans, LA, and have it in culture. Milestone Fully Met 4. Make germplasm release of root-knot nematode resistant lines with improved agronomic traits. Evaluated lines in field and greenhouse. Will write germplasm release in 2006. Milestone Substantially Met 5. Develop new linkage map of SSR and QTL in RIL lines from an intraspecific cross. Developed map with about 200 markers in a recombinant inbred population from the cross of HS-46 x MARCABUCAG8US-1-88. Will publish in 2006. Milestone Fully Met 6. Report and deposit in public database 100 EST containing SSR markers. Submitted report on 84 EST-SSR primer pairs covering more than 200 SSR markers developed from G. hirsutum EST database of GeneBank. Submitted these to Cotton Microsattelite Database organized by Cotton Incorporated. Currently working on 200 additional EST-SSR primer pairs from G. arboretum EST-database. Milestone Fully Met 7. Complete development of RIL for 4 populations from
crosses of nematode resistant x susceptible lines. RIL selfed 4 generations from individual F2 plants in 4 crosses. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006: Develop about 20 day neutral race accession lines. Make crosses and do selection. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. As industry develops new transgenic events, we expect to work with interested industries to evaluate them. Make germplasm release of random mated populations involving 4 cycles of random mating with 11 elite lines. Expect to make the release in 2006. Make germplasm release of additional chromosome substitution lines. Four new lines are under development and should be released in 2006. Report on discovery of genes for resistance to reniform nematode. Manuscripts will be written. Add
about 100 BNL SSR to intraspecific RIL linkage map. Expect to develop linkage map with 254 markers. FY 2007: Develop about 20 day neutral race accession lines. Make selections and initiate new crosses. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. As industry requests cooperation, we expect to do so. Complete development of 3 chromosome specific RIL from chromosome substitution lines. Work is underway and lines should be developed in 2007. FY 2008: Develop about 20 day neutral race accession lines. Make selections and initiate new crosses. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Work with industry as they request our assistance to evaluate new constructs. Complete development of chromosome substitution lines from G. tomentosum in G. hirsutum. Make crosses of these lines with cultivars and increase seed for germplasm release of CS-T substitution lines. Identify PCR based markers closely
associated with root-knot nematode resistance. Identify new markers in recombinant inbred lines. Report characterization of MIC gene in root-knot nematode resistant lines. Describe several genes in the gene family and determine their relationships. Compare MIC genes in US and Uzbekistan resistant lines. Complete linkage map of several kinds of PCR based markers including SNPs. Expect to have an intraspecific map of SNPs. 4a What was the single most significant accomplishment this past year? Resistance to reniform nematode found in wild G. hirsutum accessions: Resistance to reniform nematode in two wild exotic accessions which we had converted to day neutrality was identified by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with ARS at College Station, TX. Reniform nematode is a serious pest for growers of upland cotton and no good plant resistance is available in cultivars. This discovery provides genes for plant breeders to use to
develop resistant cultivars which should provide growers with an economic and environmentally friendly method of control of this destructive pest. 4b List other significant accomplishments, if any. New chromosome substitution lines of Gossypium tomentosum in G. hirsutum: We developed a series of 45 aneuploid BC0F1 substitution lines for the chromosomes and chromosome arms of Gossypium tomentosum substituted into G. hirsutum. This addresses the need for an effective way to move genes from wild tetraploid species of cotton into the cultivated species. The hypoaneuploid BC0F1 plants were identified, through cooperative research between scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Texas A & M University, based on plant phenotype, meiotic metaphase I configuration analysis of acetocarmine- stained microsporocytes, and molecular markers. These lines will be new cytogenetic resources for use in genetic mapping and when substitution lines
are developed these should potentially impact cotton genetics and breeding by broadening the germplasm base in Upland cotton through chromosome specific introgression from G. tomentosum species. Molecular mapping of natural leaf defoliant trait with SSR markers: We associated 4 SSR markers, JESPR 13, 56, 153, and 178, with a natural leaf defoliation trait in an Uzbekistan cotton line and located these markers to the short arm of chromosome 18. This addresses the lack of genetic knowledge about a natural defoliation trait bred into a cultivar of cotton in Uzbekistan. About 270 SSR primer pairs were screened against 66 individual F5 plants from the extremes in phenotypes, in an intraspecific cross of 2 Uzbekistan lines of G. hirsutum, in cooperative research among scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, The Institute of Genetics and Plant Experimental Biology Academy of Sciences of Uzbekistan, at Tashkent, Uzbekistan, and the
Department of Biology, West Virginia State University, WV. The PCR based SSR markers associated with this leaf defoliant trait has the potential to assist breeders via marker-assisted selection and to assist molecular biologists with map based cloning of this important gene. BAC-derived SSR as genome framework markers in cotton: The chromosomal association of 50 BAC derived SSR loci was determined by scientists in the Genetics and Precision Agricultural Research Unit at Mississippi State, MS, ARS scientists at Stoneville, MS, ARS scientists at College Station Texas, and Texas A&M University scientists. BAC derived SSR address the problem of integrating physical and genetic maps of the cotton genome. These PCR based markers were identified based on the deletion analysis with the two different sets of the aneuploid substitution lines derived from G. barbadense (3-79) and G. tomentosum. Chromosome based BAC-derived SSR markers will provide bridges that will help integrate physical
maps with genetic maps and will provide a useful framework of markers for chromosome based genome sequencing and facilitate positional candidate gene cloning, comparative genome analysis and as a platform in future of chromosome based genome sequencing project. Chromosomal location of a Cyclin-dependent kinase fiber gene: A SNP associated with a cyclin dependent kinase A gene was located to the long arm of chromosome 16 through cooperative research among scientists in the Geneticis and Precision Agriculture Research Unit at Mississippi State, MS, Mississippi State University and Texas A & M University. With this discovery we demonstrated that single primer extension technology could be used to identify SNP markers associated with this previously cloned and sequenced fiber gene in cotton. This addresses the problem of the need for a better understanding of the genetics of fiber development in cotton. When several fiber genes are located to chromosomes plant breeders will have a
better understanding of how to manipulate genes for fiber quality in cultivar development. Genetics of resistance to reniform nematode in G. arboretum: The genetic basis of resistance to reniform nematode in G. arboretum was determined to be multigenic and to involve partial dominance in research by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This addresses the problem of how to use these genes for resistance in the development of cultivars. This discovery implies that it will be a complex process for breeders to transfer these resistance genes from G. arboretum to upland cotton with conventional technology. New Assignment of SSR markers to chromosomes: From a screen of 397 BNL SSR markers, 142 were assigned to specific chromosomes, linkage groups A01 was assigned to chromosomes 8, A03 was assigned to chromosome 11, and SSR markers were assigned to chromosome 14, by using interspecific hyperaneuploid hybrids of G. hirsutum x G. tomentosum and G.
hirsutum x G. barbadense. This was accomplished by cooperative research between scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and scientists at Texas A&M University. This addresses the problem of a need for assignment of framework markers to all the chromosomes in cotton. These new and confirmed marker assignments will greatly accelerate the construction of useful linkage maps and the location of QTL linked to fiber, agronomic, and pest resistance genes in upland cotton. 4d Progress report. Day neutral genes bred into wild accessions of G. hirsutum: Day neutral selections in 112 F2 populations between wild accessions and cultivars, and crosses of 100 photoperiodic exotic accessions with a day neutral cultivar were made by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. This addresses the need for useful genetic diversity for cotton breeding improvement, especially genes for improved fiber quality.
These selections will provide diverse genes from exotic accessions that are useful for cotton breeding and improvement. Advanced linkage map in Gossypium hirsutum: A linkage map of 254 SSR markers polymorphic in the intraspecific recombinant inbred population from the cross of HS 46 x MARCABUCAG8USA1-88 is being developed by scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS. All current linkage maps of molecular markers have been developed using interspecific populations and, thus, are of limited use to plant breeders. This intraspecific linkage map will accelerate the discovery of QTL in upland cultivars and breeding lines that are linked to fiber, agronomic, and pest resistance traits and will facilitate marker assisted selection in cotton. Linkage between SSR markers and QTL in cotton: A linkage map of SSR loci with QTL for agronomic and fiber traits is being developed in a recombinant inbred population from the cross of HS 46 x
MARCABUCAG8USA1- 88 by scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Mississippi State University. There are no linkage maps available from intraspecific G. hirsutum populations that link SSR with QTL for fiber, agronomic, and pest resistance This map will provide valuable information for breeders using marker assisted selection for these important traits as well as provide information about the distribution of these gene loci across the genome of cotton. New transgenes for insect resistance in cotton: The widestrike gene technology was evaluated for efficacy against tobacco budworm, bollworm, and fall armyworm under a Trust Agreement between the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Dow AgroSciences. This addresses the need for additional insect transgenes by growers and the need for better ways to implement resistance management. The data are being supplied to the company and will also be
published. Cultivars with Widestrike technology were available to growers in 2005. The availability of Widestrike technology provides growers with new genes for insect control and should improve resistance management. Random mating populations for cotton breeding: A random mated population is being developed through five cycles of random mating beginning with a diallele cross among 11 elite lines. This involves cooperation among the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, Mississippi State University, North Carolina State University, and Cotton Incorporated. This addresses the problem of adverse linkages between fiber quality genes and yield genes. The first crosses were made as an 11 parent diallele using diverse parents from all areas of the US Cotton Belt. The fourth cycle of intermating was made during the winter in the nursery and the fifth cycle of crossing was made in the field in 2005. Populations of F2 plants from cycle 0, 1, 2, and 3
were evaluated in a two location yield test in 2005. Abundant recombination seems to be occurring based upon visual observations and on yield in cycles 0, 1, and 2. Fingerprinting of the 11 parental lines continues with SSR markers. These random mating populations will provide valuable diversity and new gene combinations useful to all public and private cotton breeding programs. Introgression of useful genes from G. barbadense into G. hirsutum: Germplasm with individual chromosomes from G. barbadense substituted for the corresponding chromosome in G. hirsutum are being developed by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Texas A&M University. This addresses the problem of poor success in the introgression of genes from G. barbadense into G. hirsutum using crosses at the whole genome level. Genetic data from crosses of 13 substitution lines with 5 cultivars was analyzed and manuscripts are under preparation. This
information will be valuable to industry as they use the substitution lines we released in 2004 to speed up the introgression of genes from G. barbadense into upland cultivars. Insect resistant transgenes in cotton: Several transgene constructs and events developed by Bayer Crop Science were evaluated for insect control efficacy by the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, working under a Trust Agreement with Bayer. There continues to be interest by industry in development of new transgenes for insect control as well as an interest by growers in the availability of new transgenes for insect control. Field plots are infested with H. zea and laboratory tests are conducted with tobacco budworm, cotton bollworm, and fall armyworm. Results of this research should speed the development of transgenic insect resistant cultivars of cotton by Bayer Crop Science. This should ultimately benefit growers as well as improve resistance management. PCR based
molecular markers and QTL: We identified 133 SSR-containing consensus EST sequences by analyzing 9,948 EST sequences from G. hirsutum and identified 1900 consensus and non-redundant sequences that contain an SSR motif of at least 18-bp length from 38,598 sequences of G. arboreum in an EST database. We detected polymorphism among a few selected intra and interspecific cotton genotypes and made the first report on PCR-based EST-SSR markers in cotton. This involved cooperation between the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and Alabama A&M University, via an NRI grant. More than 50% of these EST- SSRs are associated with fiber ESTs. We have submitted a report on 84 EST-SSR primer pairs covering more than 200 SSR markers from G. hirsutum EST database to Cotton Microsattelite Database at Cotton Incorporated. We are currently working on 200 additional EST-SSR primer pairs developed from G. arboretum database. We have developed a SNP detection
method in cotton. Work will continue to assign these markers to chromosomes. These results have expanded the knowledge of functional genomics in cotton. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The Genetics and Precision Agriculture Research Unit at Mississippi State, MS, in cooperation with Mississippi State University, made two germplasm releases of several high strength fiber lines in FY 2004 which can be used by industry to breed improved fiber quality cultivars. Thirty-five day neutral lines derived from exotic G. hirsutum photoperiodic germplasm have been released as germplasm for use in cultivar development. These day neutral lines from exotic G. hirsutum accessions offer new genes to breeders in lines that they can use in their breeding programs. A germplasm release was made of 17 chromosome substitution lines in which chromosomes from G. barbadense have been substituted into G. hirsutum lines. In
addition we are now writing a manuscript to show industry breeders how to use the genes in these lines for improvement of their new cultivars. Resistance to reniform nematode found in wild G. hirsutum accessions: Reniform nematode is a serious pest for growers of upland cotton and no good plant resistance is available in cultivars. Scientists in the Genetics and Precision Agriculture Research Unit at Mississippi State, MS, and ARS at College Station, TX, identified resistance to reniform nematode in two wild accessions which we had converted to day neutrality. This discovery provides genes for plant breeders to use to develop resistant cultivars which should provide an economic and environmentally friendly method for growers to use for control of this destructive pest. Numerous manuscripts have been published on new statistical and breeding analyses methodologies. Software for these analyses are available from the scientists in the Genetics and Precision Agriculture Research Unit at
Mississippi State, MS, cooperating with Mississippi State University. Twenty-four manuscripts were published in FY 2004 and 23 manuscripts in FY 2005 describing research in this project. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? New germplasm lines have been developed and released. These are immediately available for use by the first set of end users which are public scientists and scientists in commercial breeding firms. As they use these germplasms to improve cultivars, these genes in the form of improved cultivars, will become available to the final end user which is the grower. Manuscripts have been published describing new statistical methodologies for genetic analyses of plant breeding experiments. These can be used immediately by
plant breeders. These methodologies should improve plant breeders ability to identify and utilize genetic variation in their breeding lines.
Impacts
(N/A)
Publications
- Wu, J., Jenkins, J.N., McCarty Jr., J.C., Zhu, J. 2004. Genetic association of yield with its component traits in a recombinant inbred population of cotton. Euphytica. 149:171-179.
- McCarty Jr., J.C., Wu, J., Jenkins, J.N., Guo, X. 2005. Evaluating American and China cotton cultivars and their crosses for improvement. Cotton Science. 17:47-55.
- McCarty Jr., J.C., Jenkins, J.N., Wu, J. 2005. Potential of primitive accessions for cotton improvement. Mississippi Agricultural and Forestry Experiment Station Technical Bulletin 1141. 22 p.
- Saha, S., Van'T Hof, J. 2005. Cotton fiber cells are arrested at G1 stage. Journal of New Seeds. 7:1-8.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C. 2005. Distribution of boll number and lint yield by time and position in upland cotton cultivars. Applied Statistics In Agriculture Conference Proceedings. p. 296-309.
- Wu, J., Zhu, J., Jenkins, J.N., McCarty Jr., J.C. 2005. Constructing linkage maps with achiasmatic gametogenesis. Acta Genetica Sinica. 32:608- 615.
- Sakhanokho, H.F., Zipf, A., Rajasekaran, K., Saha, S., Sharma, G.C., Chee, P.W. 2004. Somatic embryo initiation and germination in diploid cotton (Gossypium arboreum L.). In Vitro Plant. 40:177-181.
- Jenkins, J.N., McCarty Jr., J.C. 2005. Effects of VIP on selected cotton insect pests in field and laboratory experiments [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1046.
- Lafoe, J.M., Jenkins, J.N., McCarty Jr., J.C., Gutierrez, O.A., Robinson, A.F. 2005. Resistance to reniform nematode in exotic cotton lines [abstract]. National Cotton Council Beltwide Cotton Conference. p.1068.
- Cash, L., Jenkins, J.N., McCarty Jr., J.C. 2005. Influence of four plant populations on boll retention and lint yield on four commercial cultivars [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1019.
- Miller, H.T., Jenkins, J.N., McCarty Jr., J.C. 2005. Growth and fruiting habits of DP 555 BG/RR in various row patterns and plant spacings [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1001.
- Wu, J., Jenkins, J.N., McCarty Jr., J.C. 2005. Mixed model based conditional analysis for complex traits [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1010.
- Gutierrez, O.A., Jenkins, J.N., McCarty Jr., J.C., Bowman, D.T., Watson, C. E. 2005. Development of breeding populations in cotton through random mating [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1000.
- Guo, Y., Saha, S., Yu, J., Jenkins, J.N., Kohel, R.J., Stelly, D.M. 2005. Chromosomal assignment of bac-derived SSR markers in cotton (Gossypium hirsutum L.) [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1067.
- Karaca, M., Saha, S., Callahan, F.E., Jenkins, J.N., Read, J.J., Percy, R. G. 2004. Molecular and cytological characterization of a cytoplasmic- specific mutant in pima cotton (Gossypium barbadense L.). Euphytica. 139:187-197.
- McCarty Jr., J.C., Jenkins, J.N. 2004. Notice of release of 14 upland cotton, Gossypium hirsutum L., primitive derived germplasm lines with improved fiber strength. Official Release of USDA, ARS and Mississippi Agricultural and Forestry Experiment Station. 7/29/04.
- McPherson, M.G., Jenkins, J.N., Watson, C.E., McCarty Jr., J.C. 2004. Inheritance of root-knot nematode resistance in M-315 RNR and M78-RNR cotton. Journal of Cotton Science. 8:154-161.
- Saha, S., Wu, J., Jenkins, J.N., McCarty Jr., J.C., Gutierrez, O.A., Stelly, D.M., Percy, R.G., Raska, D.A. 2004. Effect of chromosome substitutions from Gossypium hirsutum L. 3-79 into G. hirsutum L. TM-1 on agronomic and fiber traits. Journal of Crop Science. 8:162-169.
- Callahan, F.E., Zhang, X., Ma, D., Jenkins, J.N., Hayes, R.W., Tucker, M.L. 2004. Comparison of MIC-3 protein accummlation in response to root-knot nematode infection in cotton lines displaying a range of resistance levels. Journal of Cotton Science. 8:186-190.
- McCarty Jr., J.C., Jenkins, J.N., Wu, J. 2004. Use of primitive accessions in cotton improvement. In: Fischer, T., editor. Proceedings 4th International Crop Science Congress, September 26-October 1, 2004, Brisbane, Australia. 2004 CDROM.
- Jenkins, J.N., McCarty Jr., J.C., Wu, J., Saha, S., Stelly, D. Chromosome substitution lines from Gossypium barbadense L. as sources for G. hirsutum L. improvement. In: Fisher, T., editor. Proceedings 4th International Crop Science Congress, September 26-October 1, 2004, Brisbane, Australia. 2004 CDROM.
- McCarty Jr., J.C., Jenkins, J.N. 2004. Primitive cotton germplasm: yield and fiber traits for twenty-one day-neutral accessions. Mississippi Agricultural and Forestry Experiment Station Research Report 23(14). 6 p.
- Ulloa, M., Saha, S., Jenkins, J.N., Meredith, Jr., W.R., McCarty, Jr., J.C. , Stelly, D.M. 2005. Chromosomal assignment of RFLP linkage groups harboring important QTLs on an intraspecific cotton (Gossypium hirsutum L.) joinmap. Journal of Heredity. 96(2):1-13.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This CRIS project is assigned to National Program 301 and the component, Genetic Characterization, Manipulation, and Genetic Improvement of Cotton. It also contributes solutions to National Program 302 and the component, Plant Defense and Secondary Metabolism and National Program 304 and the component, Development of New and Improved Pest Control Technologies and Integration of Component Technologies for IPM Systems. Objective 1: Broaden the genetic base for upland cotton improvement. Objective 2: Develop pest resistant germplasm, breeding methodologies and marker assisted selection. Objective 3: Identify genes and gene products associated with resistance to nematodes. Objective 4: Develop and use PCR based DNA markers for improvement of upland cotton. The approaches are: Approach 1: Broaden
the germplasm base of upland cotton and develop special breeding populations by (a) accessing genetic diversity in exotic photoperiodic accessions, (b) developing elite breeding populations through random mating, (c) developing backcrossed chromosome substitution lines, and (d) developing chromosome specific recombinant inbred lines for selected chromosome substitution lines. Approach 2: Develop pest resistant germplasm, breeding methodologies, and agronomic and fiber evaluations by (a) evaluating lines for agronomic and fiber properties and genetic analyses, (b) evaluating lines for tobacco budworm resistance, (c) evaluating industry developed transgenic insect resistant lines for performance of specific transgenes and transformation events, (d) discovery and development of root-knot and reniform nematode resistant germplasm, (e) development of breeding methodologies for root- knot nematode resistance, and (f) conduct evaluations of breeding lines in a regional root-knot nematode
nursery. Approach 3: Identify genes and gene products correlated with resistance to nematodes by (a) developing hairy root system in cotton, and (b) characterizing MIC genes. Approach 4: Development and utilization of PCR based DNA markers for improvement of upland cotton by (a) developing informative PCR based EST- SSR markers, (b) developing SNP markers specific to known functional and fiber associated genes, (c) assigning EST-SSR and SNP markers to chromosomes, (d) linking EST-SSR and QTL for agronomic and fiber traits, and (e) linking molecular markers with genes for nematode resistance. Cotton growers are experiencing shrinking profits in cotton production. This is due largely to higher input costs without increases in yield or quality; thus, many producers are having a difficult time continuing to produce cotton profitably. Diversity among varieties and breeding programs needs to be expanded because breeders depend upon diversity to breed new and improved varieties. Yields
of cotton have been on a plateau for the past 10 years. Research shows that all current varieties are closely related. Tobacco budworm, cotton bollworm, and nematodes are the most serious pests of cotton in the U.S. Control costs for insects range from $60 to $90 per acre. Insects are resistant to many insecticides. There are no commercial varieties resistant to reniform nematode and none with a high level of resistance to root-knot nematode; however, the level of root-knot nematode resistance is increasing in commercial cultivars. Our research program has discovered and developed germplasm with a very high level of resistance to root-knot nematode; however, there is not a good selection method for use in applied breeding. Molecular markers linked with nematode resistance should make breeding resistant varieties feasible. Very little is known about the genomic location of important genes for agronomic, fiber, and pest resistance. Knowledge is limited about the molecular biology
of cotton. This area needs expanded research. This is a critical time in cotton production, as costs are escalating, pests are developing increased levels of resistance to pesticides, nematode pests are increasing, and prices growers receive continue to be low. The genetic base that cotton breeders use in applied breeding programs is narrow. The genetic base in the genus Gossypium is large, but greatly limited in its usefulness to applied breeders, because of photoperiodicity in exotic G. hirsutum accessions, cross incompatibility problems associated with interspecific crosses, and the lack of molecular knowledge and/or markers in cotton that allows breeders to combine conventional and molecular marker approaches in breeding. Thus, diversity exists in the genus, but requires discovery, early stage breeding, linkage of DNA markers with QTL and pest resistance genes in order to make the needed advances in development of new varieties with enhanced pest resistance, improved fiber,
and increased yield. DNA markers linked to agronomic traits should significantly increase the efficiency of various breeding strategies via marker assisted selection. The two major limiting factors in the use of molecular markers in cotton are 1) limited number of markers available in the public sector and 2) lack of marker association with economically important QTL in cotton. The tetraploid species, G. barbadense, G. tomentosum, and G. mustelinum, are reservoirs of important genes for pest and disease resistance, and for improved agronomic and fiber traits. Breeders face many challenges when introgressing genes from these species. An alternative approach to conventional introgression of alien genes for improved traits into an Upland background is via chromosome substitution lines. New transgenes and new natural genes are needed for pest resistance and scientists in this CRIS project will seek to discover new genes for pest resistance. Nematode pests reduce profits for cotton
growers and are implicated as a major factor in the yield plateau observed over the past ten years. The National Cotton Council estimated a loss in yield equivalent to about $250 million per year due to nematode pathogenesis. Profits are lost through money spent for conventional pesticide control and through yield losses from failure to completely control nematode populations. Plant genes for resistance to these pests should offer an effective control approach. Genes for nematode resistance exist in Gossypium, but breeding improved cotton varieties with nematode resistance is difficult due to labor intensive, time consuming bioassays required to identify resistant progeny. Molecular approaches offer a way to improve the speed of development in breeding programs through marker assisted selection and identification of resistance genes for genetic engineering. To date no genes for root-knot nematode or reniform nematode resistance in cotton have been isolated and sequenced. The
work proposed here characterizing gene expression during early interaction of roots with nematodes should provide useful tools to aid the incorporation and use of resistance genes for control of nematodes in cotton. New transgenes for pest resistance are being developed by several commercial companies. As industry develops these transgenes we will cooperate with companies that ask us to do so. We presently have trust agreements with three companies. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004): Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of chromosome substitution lines of G. barbadense in G. hirsutum. Year 2 (FY 2005): Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Develop
hairy root system in cotton for nematode research. Make germplasm release of root-knot nematode resistant lines with improved agronomic traits. Develop new linkage map of SSR and QTL in RIL lines from an intraspecific cross. Report and deposit in public database 100 EST's containing SSR markers. Complete development of RIL for 4 populations from crosses of nematode resistant x susceptible lines. Year 3 (FY 2006): Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of random mated populations involving 4 cycles of random mating with 11 elite lines. Make germplasm release of additional chromosome substitution lines. Report on discovery of genes for resistance to reniform nematode. Add about 100 BNL SSR's to intraspecific RIL linkage map. Year 4 (FY 2007): Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new
transgenic constructs from industry for resistance to Lepidoptera insects. Complete development of 3 chromosome specific RIL from chromosome substitution lines. Year 5 (FY 2008): Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Complete development of chromosome substitution lines from G. tomentosum in G. hirsutum. Identify PCR based markers closely associated with root-knot nematode resistance. Report characterization of MIC gene in root-knot nematode resistant lines. Complete linkage map of several kinds of PCR based markers including SNP's. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled for completion in Year
1 (FY 2004). Develop and make germplasm release of about 20 day neutral race accession lines. Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. Make germplasm release of chromosome substitution lines of G. barbadense in G. hirsutum. We fully met all three milestones for FY 2004. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2 (FY 2005): Develop and make germplasm release of about 20 day neutral race accession lines. Each year we will evaluate 100 F2 populations and make germplasm release of about 20 day neutral lines. It takes 10 years to develop a BC4 day neutral line from exotic germplasm because of photoperiodicity requiring certain crosses to be made in the short day winter nursery. We start about 20 new lines into the scheme each year and release about 20 lines as day neutral germplasm.
Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects. We will work each year with Bayer, Syngenta, and Dow to evaluate their new insect resistant transgenic constructs for efficacy for Lepidoptera control. Develop hairy root system in cotton for nematode research. We will hire a molecular geneticist to fill a vacant position and develop the hairy root system for nematode research. Make germplasm release of several root-knot nematode resistant lines with improved agronomic traits. These lines will undergo the final stages of evaluation and yield testing in 2004 and 2005. Germplasm release expected in 2005. Develop new linkage map of SSR and QTL in RIL lines from an intraspecific cross. We have already developed the RIL population, collected samples for DNA, and evaluated for QTL in field plots. The SSR markers will be run in 2005 to make the new linkage map. Report and deposit in public database 100 EST's containing SSR markers. Markers have been
confirmed and information will be assembled and entered into database in 2005. Year 3 (FY 2006): Complete development of RIL for 4 populations from crosses of nematode resistant x susceptible lines. In FY 2005 two generations will be grown and self pollinated and this will allow completion of RIL lines in 2005. Develop and make germplasm release of about 20 day neutral race accession lines (see Year 2, FY 2005 for details). Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects (see Year 2, FY 2005 for details). Make germplasm release of random mated populations involving 4 cycles of random mating with 11 elite lines. We are presently in Cycle 3 and cycle 4 should be completed in 2004, lines will be evaluated in FY 2005 and released as germplasm in 2006. Report on progress of discovery of genes for resistance to reniform nematode. We have germplasm that we believe will provide resistance genes. By 2006 we should have confirming data from greenhouse
evaluations. Expect to report on this in 2006. Add about 100 BNL SSR's to intraspecific RIL linkage map. The RIL population DNA has been collected and by 2006 we should be finished with the genotyping and with linkage map development. Year 4 (FY 2007): Develop and make germplasm release of about 20 day neutral race accession lines (see Year 2, FY 2005 for details). Evaluate new transgenic constructs from industry for resistance to Lepidoptera insects (see Year 2, FY 2005 for details). Complete development of 3 chromosome specific RIL from chromosome substitution lines. We are growing the F3 in 2004 and are making self pollinated generations in the greenhouse at the rate of 2-3 per year. We should have the RIL by 2007. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004: The genetic base for upland cotton is narrow and diverse germplasm is needed to provide genes for agronomic, fiber, and pest resistance
improvement. The Genetics and Precision Agriculture Research Unit of the Crop Science Research Laboratory made a germplasm release of 21 day neutral exotic lines that have genes for improvement in fiber strength and perhaps other traits. The Research Unit also cooperated with David Stelly of Texas A&M University at College Station, TX, to make a germplasm release of 17 chromosome substitution lines that have specific G. barbadense chromosomes substituted into G. hirsutum to replace the corresponding chromosome. The release of day neutral exotic lines provides industry with new and diverse genes for cultivar improvement and the release of the chromosome substitution lines offer a new way to introgress G. barbadense genes into upland cultivars. B. Other Significant Accomplishment(s), if any: None. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress report: 1. Reniform nematode is a major pest in cotton and no cultivars are
resistant. The Genetics and Precision Agriculture Research Unit of the Crop Science Research Laboratory in cooperation with Forrest Robinson from ARS at College Station, TX, began a breeding program with exotic germplasm that is resistant to reniform nematode. We obtained exotic resistant plants and made crosses to extract genes in improved germplasm. This should provide resistance genes for commercial use in resistant cultivar development. 2. Reniform nematode is a major pest in cotton and no cultivars are resistant. The Genetics and Precision Agriculture Research Unit of the Crop Science Research Laboratory confirmed resistance in several exotic G. arboretum germplasms, made crosses between resistant and susceptible lines, and evaluated 3 F2 populations. Resistance genes are segregating in this material. This species is a diploid and it may be possible to clone the resistance gene(s) and transform into G. hirsutum. 3. Reniform nematode continues to be a major problem in
cotton production. The Genetics and Precision Agriculture Research Unit in cooperation with Mississippi State University, Mississippi Agricultural and Forestry Experiment Station selected parental lines and these have been evaluated in the greenhouse. F2 populations with G. arboretum have been evaluated in greenhouse and resistance is segregating. Resistance appears to be due to one or more recessive genes. Additional F2 populations are being evaluated. 4. Fiber length and strength need to be improved in cotton. The Genetics and Precision Agriculture Research Unit in cooperation with Dr. Govind Sharma of Alabama A&M, an 1890 University, are working on this. Several visits to our laboratory were made by Alabama A&M scientists during the year. Research is under way to seek fiber specific genes on chromosome substitution lines CS-B22Lo and CS-B22sh. We are growing plant materials in the field for sampling specific aged bolls. A SCEP employee with ARS is located at Alabama A&M and
is working on the chromosome substitution lines. Research continues at Alabama A&M on reniform nematodes. Due to the death of Dr. Al Zipf of Alabama A&M, a new scientist has been hired for the nematode part of this project. 5. To conduct plant resistance research for insect resistance, a laboratory colony of tobacco budworm, cotton bollworm, and fall armyworm must be maintained. The Genetics and Precision Agriculture Research Unit in cooperation with Mississippi State University, Mississippi Agriculural and Forestry Experiment Station maintain this colony. ARS does not have an Entomologist assigned to the research; however, we grow laboratory colonies of insects to use in evaluating cotton lines for resistance to these pests. The Entomology Department at Mississippi State University provides an entomologist with experience in rearing insects to advise us in our small scale rearing program. Excellent quality and quantities of bollworm, tobacco budworm, and southern fall armyworm
were reared and utilized in field plot and laboratory research in FY 2004. Advice from the Mississippi State Entomologist was very valuable to research in the parent CRIS project. 6. The currently available Bt genes for resistance to lepidopteran pests of cotton are Bollgard and Bollgard II from Monsanto. The Genetics and Precision Agriculture Research Unit under a Trust Agreement with Syngenta Cooperation evaluated some of their resistant lines with genes from the vegetative phase of the Bt organism. These are called VIP genes. The VIP toxin is produced during the vegetative phase of growth of the bacteria Bacillus thuringiensis. We evaluated their VIP gene(s) in various experimental lines for efficacy of control of tobacco budworm and cotton bollworm. If successful, this research would offer new genes in transgenic cotton for insect control and should improve resistance management strategies. Several transgenic lines were evaluated in 2004 for efficacy of control of
lepidopteran insects. 7. To widen the germplasm base of transgenes for insect resistance the Genetics and Precision Agriculture Research Unit conducted research under a Trust Agreement with Dow Agro Sciences. New Bt gene constructions were evaluated for efficacy against tobacco budworm and cotton bollworm. Data are being supplied to the company. Results are favorable for use of these transgenes for insect resistance. Dow has announced that its transgene, Widestrike, should be available to growers in 2005. We evaluated several experimental lines in FY 2004 for growth of tobacco budworm, cotton bollworm, and fall armyworm on leaf and boll tissue in the laboratory. 8. To broaden the genetic base of breeding populations for cotton improvement, the Genetics and Precision Agriculture Research Unit, via a Reimbursable Cooperative Agreement with Cotton Incorporated and in cooperation with Dr. Clarence Watson of Mississippi State University and Dr. Daryl Bowman of North Carolina State
University are developing a random mated population from four cycles of random mating beginning with a diallele cross among 11 elite lines. The first crosses were made as an 11 parent diallel using diverse parents from all areas of the U.S. Cotton Belt. The third cycle of intermating was made during the winter in the nursery and the fourth cycle of crossing was made in the field in 2004. Populations of F2 plants from cycle 0, 1, and 2 were evaluated in a two location yield test in 2004. Abundant recombination seems to be occurring based upon visual observations and on yield in cycles 0 and 1. Fingerprinting of the 11 parental lines continues with SSR markers. These random mating populations will provide valuable diversity and new gene combinations useful to all public and private cotton breeding programs. 9. Introgression and use of genes from G. barbadense into G. hirsutum is not very successful at the whole genome level. The use of chromosome substitution lines can greatly
improve this process. The Genetics and Precision Agriculture Research Unit in cooperation with Dr. David Stelly of Texas A&M University are developing germplasm with an individual chromosome from G. barbadense substituted for the corresponding chromosome in G. hirsutum. Seventeen chromosome substitution lines were released as germplasm in 2004. These should speed up the introgression of genes from G. barbadense into upland culrivars. 10. There continues to be interest by industry in development of new transgenes for insect resistance. The Genetics and Precision Agriculture Research Unit working under a Trust Agreement with Bayer is evaluating Bayer's version of transgenes for insect resistance. We evaluated several experimental lines from Bayer in 2004. Field plots are infested with H. zea and laboratory tests are conducted with tobacco budworm, cotton bollworm, and fall armyworm. 11. There continues to be a shortage of PCR based molecular markers in cotton linked with QTL for
important genes. The Genetics and Precision Agriculture Research Unit is working under a Reimbursable Agreement with Alabama A&M University under an NRI grant. The objectives are to discover EST-SSR and SNP markers associated with fiber genes and identify their chromosomal locations. 133 SSR-containing consensus EST sequences were identified by analyzing 9,948 EST sequences from G. hirsutum. Primers were designed for 84 and tested for polymorphism among four cotton lines belonging to G. hirsutum and G. barbadense. About 1900 consensus and non-redundant sequences contain an SSR motif of at least 18- bp length from 38,598 sequences of G. arboreum in an EST database. We constructed 206 primer pairs from these non-redundant sequences and detected polymorphism among a few selected intra and interspecific cotton genotypes. This is the first report on PCR-based EST SSR markers in cotton. More than 50% of these EST SSRs are associated with fiber EST's. We are making progress in
development of a method for detection of SNP's in cotton. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The two germplasm releases we made in FY 2004 contain genes that can be used by breeders to improve new cultivars. The day neutral lines from exotic accessions offer completely new genes to breeders. The chromosome substitution lines offer an improved way to introgress genes from G. barbadense into G. hirsutum cultivars and breeding lines. We developed a way to use the chromosome substitution lines to probe the useful genes on specific chromosomes in any cultivar. A manuscript is being prepared on this technique at this time. Specific agronomic and fiber traits with chromosomes using chromosome substitution lines have been identified. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other
scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We made a germplasm release of 21 day neutral lines of cotton developed from exotic accessions. Many had genes for improved fiber strength. These have gone to public and private breeders for use in cultivar development. We made a germplasm release in cooperation with Texas A&M University of 17 chromosome substitution lines that have specific chromosomes from G. barbadense substituted into G. hirsutum. These should make it easier for breeders to introgress genes from G. barbadense into cultivars compared to making an interspecific cross of G. hirsutum x G. barbadense. There are no known constraints to using genes from released lines to improve cultivars. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Saha, S., Jenkins, J.N., McCarty, J.C., Wu, J., Gutierrez, O.A., Cantrell, R.G., Percy,
R.G., Stelly, D.M. 2004. Chromosome substitution lines: Important genomic resources in cotton. Memorial Symposium, 4 June 2004, Alabama A&M University.
Impacts
(N/A)
Publications
- McCarty Jr., J.C., Jenkins, J.N., Wu, J. 2004. Primitive accession derived germplasm by cultivar crosses as sources for cotton improvement: I. Phenotypic values and variance components. Crop Science. 44:1226-1230.
- McCarty Jr., J.C., Jenkins, J.N., Wu, J. 2004. Primitive accession derived germplasm by cultivar crosses as sources for cotton improvement: II. Genetic effects and genotypic values. Crop Science. 44:1231-1235.
- Wu, J., Jenkins, J.N., Zhu, J., McCarty, J.C., Watson, C.E. 2003. Monte carlo simulations on marker grouping and ordering. Theoretical Applied Genetics. 107:568-573.
- Wu, J., Jenkins, J.N., Zhu, J., McCarty, J.C., Watson, C.E. 2003. Comparisons of quantitative trait locus mapping properties between two methods of recombinant inbred line development. Euphytica. 132:159-166.
- Tan, H., Callahan, F.E., Zhang, X.D., Karaca, M., Saha, S., Jenkins, J.N., Creech, R.G., Ma, D.P. 2003. Identification of resistance gene analogs in cotton (Gossypium hirsutum L.) Euphytica. 134:1-7.
- Bowman, D.T., Gutierrez, O.A. 2003. Sources of fiber strength in the U.S. cotton crop from 1980-2000. Journal of Cotton Science. 7:164-169.
- Bezawada, C., Saha, S., Jenkins, J.N., Creech, R.C., McCarty, J.C. 2003. SSR marker(s) associated with root-knot nematode resistance gene(s) in cotton. Journal of Cotton Science. 7:179-184.
- Wu, J., Zhu, J., Jenkins, J.N. 2003. Mixed linear model approaches for quantitative genetic models. In: Kang, M.S., editor. Handbook of Formulas and Software for Plant Geneticists and Breeders. Binghamton, NY: The Haworth Press, Inc. p. 171-180.
- Jenkins, J.N. 2004. Genetically engineered crops with resistance to insects. In: Encyclopedia of Plant and Crop Science. New York, NY: Marcel Dekker. p. 506-508.
- McCarty Jr., J.C., Jenkins, J.N. 2004. Notice of release of 21 Bc4f4 noncommercial flowering day neutral germplasm lines of upland cotton involving Gossypium hirsutum L. race accessions. Germplasm Release. Official Release of USDA, ARS and Mississippi Agricultural and Forestry Experiment Station, 3/22/04.
- Stelly, D.M., Raska, W., Jenkins, J.N., McCarty, J.C., Gutierrez, O.A. Notice of release of 17 germplasm lines of upland (Gossypium hirsutum) cotton, each with a different pair of G. barbadense chromosomes or arms substituted for the respective G. hirsutum chromosomes or arms. Official Release of USDA, ARS and Mississippi Agricultural and Forestry Experiment Station, 5/04/04.
- Jenkins, J.N., Wu, J., McCarty Jr, J.C., Reddy, U., Zhu, J. 2004. A recombinant inbred population of cotton for QTL and DNA marker association. World Cotton Research Conference Proceedings. p. 352.
- Wu, J., Jenkins, J.N., McCarty Jr, J.C. 2004. Comparing two methods in evaluation of boll retentionm [abstract]. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1118.
- Ynturi, P., Jenkins, J.N., McCarty Jr, J.C., Saha, S. 2004. Segregation for root-knot nematode resistance gene(s)in F2 populations from resistant x susceptible crosses [abstract]. National Cotton Council Beltwide Cotton Conference. p. 1112.
- Miller, H.T., Jenkins, J.N., McCarty Jr, J.C. 2004. Growth and fruiting habits of DP 555 BG/RR in various row patterns and plant spacings [abstract]. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1117.
- McCarty Jr, J.C., Jenkins, J.N., Wu, J. 2004. Primitive accessions of cotton as genetic sources for improving yield and fiber properties. World Cotton Research Conference Proceedings. p. 114-118.
- Stelly, D.M., Saha, S., Raska, D.A., Jenkins, J.N., McCarty Jr, J.C., Gutierrez, O.A. 2004. Notice of release of 17 germplasm lines of upland (Gossypium hirsutum), each with a different pair of G. barbadense chromosomes or arms substituted for the respective G. hirsutum chromosomes or arms. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1205-1207.
- Saha, S., Raska, D.A., Jenkins, J.N., McCarty Jr, J.C., Gutierrez, O.A., Percy, R.G., Cantrell, R.G., Wu, J., Zhu, J., Stelly, D.M. 2004. Effect of chromosome on important quantitative traits of agronomic and fiber traits using Gossypium barbadense chromosome-specific recombinant lines of Gossypium hirsutum. World Cotton Research Conference Proceedings. p. 170- 174.
- Gutierrez, O.A., Jenkins, J.N., McCarty Jr, J.C., Bowman, D.T., Watson, C. E. 2004. Genetic distance among cotton cultivars used in the development of random mating populations based on SSR markers and coefficient of parentage [abstract]. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1061.
- Stelly, D., Saha, S. 2004. Organizing cotton genomics through physical mapping [abstract]. Proceedings National Cotton Council Beltwide Cotton Conference. p.172.
- Abdurakhmonov, I.Y., Abdullaev, A.A., Buriev, Z.T., Rizaeva, S.M., Abdullaev, A.A., Abdukarimov, A.A., Saha, S., Reddy, U.K. 2004. Molecular mapping of natural leaf defoliation loci of cotton using SSR markers [abstract]. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1202.
- Abdurakhmonov, I., Buriev, Z., Saha, S., Musaev, J., Almatov, A., Pepper, A.E., Reddy, O.U., Jenkins, J.N., Abdullaev, A., Abdukarimov, A. 2004. Molecular tagging of fiber yield genes using intraspecific R1 lines developed in Uzbekistan. World Cotton Research Conference Proceedings. p. 320-326.
- Abdurakhmonov, I.Y., Abdullaev, A.A., Rizaeva, S.M., Buriev, Z.T., Adylova, A.T., Abdukarimov, A.A., Saha, S., Kohel, R.J., Pepper, A.E. 2004. Evaluation of G. hirsutum exotic accessions from Uzbek cotton germplasm collection for further molecular mapping purposes. Proceedings National Cotton Council Beltwide Cotton Conference. p. 1133-1142.
- Qureshi, S.N., Saha, S., Kantety, R.V., Jenkins, J.N. 2004. EST-SSR: A new class of genetic markers in cotton. Journal of Cotton Science. 8:112-123.
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